Pathology of the Surfactant System of the Mature Lung
Second San Diego Conference

ROGER G. SPRAGG and JAMES F. LEWIS

Department of Medicine, University of California, San Diego, and Medicine Service, San Diego Veterans Affairs Healthcare System, San Diego, California; and Department of Medicine, Lawson Research Institute, St. Joseph's Health Centre, University of Western Ontario, London, Ontario, Canada

	INTRODUCTION

TOP INTRODUCTION STRUCTURE/FUNCTION OF THE... SURFACTANT METABOLISM SURFACTANT-ASSOCIATED PROTEINS SURFACTANT AND HOST DEFENSES SURFACTANT IN LUNG DISEASE SUMMARY REFERENCES

The second San Diego symposium, "Pathology of the Surfactant System of the Mature Lung," held in October 1999, brought together investigators from diverse fields to discuss the remarkable recent progress made in understanding the structure, organization, and function of lung surfactant and of its constituent molecules. Concepts highlighted at the conference are noted in this report; a complete summary is published elsewhere (1).

	STRUCTURE/FUNCTION OF THE SURFACTANT FILM

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The surfactant film varies from 2 to 7 or more lamellae. Compression of this film results in a "collapsed phase" in which surfactant components are lost from the interface; subsequent reexpansion replenishes the surface layer, implicating the presence of a surfactant reservoir. Movement of lipid out of the surfactant reservoir onto the surface is enhanced by the presence of surfactant apoprotein B (SP-B). This surfactant-associated protein also increases the stability and lateral strength of the phospholipid layer by forming a helical disposition within the lipid. SP-A causes lipid aggregation, and together with SP-B facilitates tubular myelin formation.

	SURFACTANT METABOLISM

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Rat alveolar type II cells grown for 1 wk lose expression of SP-C messenger RNA (mRNA) and resemble type I cells. They regain morphologic characteristics consistent with type II cells, however, when returned to appropriate cell culture environments. The phenotype of cultured type II cells may therefore not necessarily represent that of terminally differentiated type I cells. Certain growth factors, such as keratinocyte growth factor (KGF), may enhance proliferation of rat type II cells, as well as the expression and secretion of SP-A, -B, and -D. KGF overexpression results in extensive type II cell hyperplasia and protection from injury induced by various insults (2).

An intracellular phospholipase A2 enzyme, aiPLA₂, which is maximally active in the lamellar and lysosomal compartments, serves to hydrolyze intracellular dipalmitoylphosphatidylcholine (DPPC) and is regulated, in part, by SP-A. This enzyme may play an important role in various inflammatory lung disorders.

Surfactant secreted from type II cells is adsorbed to the air-liquid interface and subsequently is converted in the presence of a serine protease belonging to a family of carboxyesterases into poorly functioning small vesicular forms (3). The dynamic change in alveolar surface area associated with respiration influences aggregate conversion, suggesting that artificial mechanical ventilation may specifically affect the endogenous surfactant system and contribute to the lung dysfunction associated with the acute respiratory distress syndrome (ARDS).

Animal models of altered surfactant metabolism include mice deficient in granulocyte-macrophage colony-stimulating factor (GM-CSF) and mice that overexpress interleukin-4. In these models alveolar and tissue pools of DPPC are increased as are concentrations of SP-A, -B, -C, and -D. These changes are likely due to a combination of increased synthesis or delayed catabolism of surfactant components, as well as abnormal macrophage function. Restoring pulmonary GM-CSF function in the knockout mice partially corrects the surfactant abnormalities, implicating GM-CSF in pulmonary disorders such as pulmonary alveolar proteinosis (4). Indeed, a clinical study involving the administration of exogenous GM-CSF to patients with alveolar proteinosis has reported promising results.

	SURFACTANT-ASSOCIATED PROTEINS

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The expression of surfactant-associated proteins may be influenced by modulation of gene transcription or mRNA stability, as well as by alterations in cell differentiation (5). Understanding these processes may be important in lung injury and repair and subsequently lead to exciting new therapeutic interventions for a variety of lung diseases.

SP-A-deficient mice (SP-A^/) have normal lung histology, alveolar disaturated phosphatidylcholine (DSPC) pool sizes, and concentrations of SP-B, -C, and -D. The tubular myelin of these animals is markedly reduced, however, and their surfactant is less resistant to serum protein inhibition compared with the wild type (6). Overexpression of SP-A results in normal lung compliance and surfactant pool sizes, and an increased resistance to protein inhibition (7). A mutant strain of mice lacking the collagenlike domain of SP-A (G8-P80) has surfactant with impaired surface tension lowering activity. Reconstitution of tubular myelin is not achieved in the SP-A^/,G8-P80 mice, whereas it is in the SP-A^{/, rat SP-A} mouse, implicating the importance of the G8-P80 residue for that function.

Infants lacking SP-B rapidly develop neonatal respiratory failure, and SP-B-deficient mice have surfactant lacking hysteresis and abnormal lamellar bodies. SP-C processing is also altered, as pro-SP-C intermediates accumulate in these mice. When a nondimerizing human SP-B mutant is expressed in the SP-B^/ background, reversal of neonatal lethality occurs, and normal levels of mature SP-C, lamellar body structure, and surfactant hysteresis are present (8). On the other hand, other mutations interrupting the internal sulfhydryl bridging of SP-B impair survival when expressed in the wild-type background, suggesting that the mutant protein might bind and trap the wild-type protein in the endoplasmic reticulum. This exemplifies how a mutation in one allele can effect expression of the wild-type allele.

A gene-targeted mouse lacking exon 2 of the single SP-C gene has no detectable SP-C in lavage fluid, but normal levels of SP-B message and protein. These mice develop normally, have normal lipid synthesis and pool sizes, and normal surfactant biophysical function. These findings suggest that at least one of the two hydrophobic surfactant proteins is necessary for survival.

SP-D-deficient mice (SP-D^/) have markedly increased tissue and alveolar phospholipid pools, a modest decrease in SP-A mRNA and protein, and normal SP-B and SP-C mRNA and protein. Incorporation of precursors into phospholipid is normal, lipid secretion is increased, and catabolism is decreased, thereby accounting for the increased phospholipid pool sizes in these animals (9). Other findings include increased conversion of large aggregates to small aggregates, patchy accumulation of large foamy alveolar macrophages, and hypertrophic type II cells with large lamellar bodies. These mice appear healthy and free of infection for up to 1 yr of age. An unexpected finding in these animals is a peribronchial monocytic infiltration. Overexpression of SP-D results in normal concentrations of SP-A, -B, and -C and of alveolar DSPC. Furthermore, endogenous SP-D expression in the overexpressing mice is also unchanged and macrophage morphology is normal.

	SURFACTANT AND HOST DEFENSES

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Although surfactant lipids appear to have an immunosuppressive role, the host's responses to insults may be dependent on the relative proportions of these phospholipids within the air space (10). The surfactant-associated proteins SP-A and SP-D bind to a variety of pathogens, enhance uptake by macrophages, and can influence the production and secretion of inflammatory cytokines and superoxide radicals from inflammatory cells (11). Specific mutations of the surfactant proteins may impair lipopolysaccharide (LPS) binding to inflammatory cells, thereby influencing the function of LPS. Finally, rats exposed in vivo to aerosolized LPS demonstrate LPS scavenging by endogenous SP-D with formation of large LPS-SP-D aggregates that are subsequently delivered to alveolar macrophages (12). It is possible, therefore, that surfactant lectins may be engineered in the future to enhance recognition of LPS and consequently influence the host's response to various infections.

Studies in which various bacteria and viruses are administered intratracheally to SP-A^/ animals show decreased microbial or viral clearance from the alveolar space, an enhanced pulmonary inflammatory reaction, and an increased release of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-), interleukin-6 (IL-6), and macrophage inflammatory protein 2 (MIP-2) into the airway. These findings are partially reversed with administration of exogenous SP-A (13).

Nitration of SP-A results in decreased adherence and subsequent clearance of Pneumocystis organisms from alveolar macrophages, although direct binding of SP-A to alveolar macrophages is not affected by nitration (14).

SP-D^/ mice have no impairment of bacterial clearance, and their alveolar macrophages increase superoxide release in contrast to the SP-A^/ mice. The surfactant proteins therefore appear to be distinct in their effects on the clearance of various organisms from the air space.

The relationship of cytokines and surfactant has also been studied. Exposure of alveolar type II cells to TNF- results in reduced cytidine triphosphate (CTP): phosphocholine cytidyltransferase (CT) activity and inhibition of DPPC synthesis. Cytokines have also been shown to regulate apoptosis of type II cells, a process that may be critical for tissue remodeling in response to inflammation. Finally, increased alveolar TNF- and IL-6 concentrations accompany alterations in the surfactant system in an ex vivo model of lung injury, suggesting a significant relationship between cytokines and surfactant alterations in acute lung injury.

	SURFACTANT IN LUNG DISEASE

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Observations

Genetic polymorphisms of the SP-B gene may represent a susceptibility factor for lung diseases such as ARDS and chronic obstructive pulmonary disease (COPD). There may also be a variant locus within intron 4 of SP-B in individuals with either small cell or non-small cell lung cancer.

In patients with ARDS, phosphatidylcholine and phosphatidylglycerol concentrations are decreased as are SP-A, -B, and -C levels. The relative proportion of functionally active large aggregate forms to small aggregates is also decreased, and surface tension measurements of surfactant isolated from these patients are abnormal.

Mechanisms Responsible for Surfactant Dysfunction

Alveolar surfactant may be inhibited by plasma proteins present within the air space of the injured lung, although this inhibition may be overcome by the addition of the nonionic polymer polyethylene glycol (PEG) to an exogenous surfactant preparation. Extracellular secretory phospholipase A₂ (PLA₂) may hydrolyze surfactant phospholipids and also increase the conversion of large aggregates into small aggregates. Hyperoxia decreases phosphatidylcholine synthesis through depletion of type II intracellular ATP concentrations, and this depletion may result from activation of poly-ADP-ribose polymerase. The combination of superoxide radicals and nitric oxide results in the formation of peroxynitrite, which can react with tyrosine residues of SP-A, rendering this protein dysfunctional.

It is apparent, therefore, that in the acutely injured lung, multiple mechanisms may contribute to surfactant dysfunction, some of which may be amenable to novel therapeutic approaches.

Exogenous Surfactant Administration

Synthetic exogenous surfactant preparations composed of lipids and either recombinant modified human SP-C (Venticute) or an SP-B-like peptide (KL₄) are more resistant to meconium inhibition when compared with modified natural preparations. Moreover, both products have shown promising results when tested in clinical trials. A natural surfactant preparation containing SP-A was superior to these products in an animal study, providing rationale for further study of the therapeutic effect of SP-A supplementation.

Partial liquid ventilation is also being tested in patients with severe ARDS. Administration of perfluorocarbon has been shown to result in a modest increase in surfactant synthesis and secretion when measured in vivo, and the combination of perfluorocarbon and surfactant was superior to surfactant administration alone in an animal model of lung injury.

Interestingly, recent demonstration of surfactant alterations very early in the course of acute lung injury provides a rationale for administering exogenous surfactant as a prophylactic agent in an attempt to prevent disease progression. Future studies will determine the efficacy of this approach.

	SUMMARY

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Knowledge of the surfactant system has grown immensely in the past decade. A variety of investigative strategies, including manipulation of surfactant protein gene expression in mice, has contributed dramatically to our understanding of the role of surfactant components in lung function. These approaches have fostered investigations that will further our knowledge of the role of lung surfactant in host defense and will provide information that should lead to improved strategies for the treatment of lung disease.

	Footnotes

Correspondence and requests for reprints should be addressed to Roger G. Spragg, M.D., San Diego VA Medical Center, 3350 La Jolla Village Drive, San Diego, CA 92161. E-mail: rspragg{at}ucsd.edu

(Received in original form April 6, 2000 and in revised form August 30, 2000).

Acknowledgments: Supported by Grant 1 R 13 HL62421-01 from the National Heart, Lung, and Blood Institute, NIH, and unrestricted educational grants from Byk Gulden, Inc., Chiesi Pharmaceuticals, Inc., Discovery Laboratories, Inc., Merck & Co., Inc., and Ross Products Division of Abbott Laboratories.

	References

TOP INTRODUCTION STRUCTURE/FUNCTION OF THE... SURFACTANT METABOLISM SURFACTANT-ASSOCIATED PROTEINS SURFACTANT AND HOST DEFENSES SURFACTANT IN LUNG DISEASE SUMMARY REFERENCES

1. Spragg RG, Lewis JF. Pathology of the surfactant system of the mature lung: 2nd San Diego Conference. Appl Cardiopulm Pathophysiol 2000; 98: 177-185 .

2. Fehrenbach H, Kasper M, Tschernig T, Pan T, Schuh D, Shannon JM, Muller M, Mason RJ. Keratinocyte growth factor-induced hyperplasia of rat alveolar type II cells in vivo is resolved by differentiation into type I cells and by apoptosis. Eur Respir J 1999; 14: 534-544 [Abstract].

3. Veldhuizen RA, Yao LJ, Lewis JF. An examination of the different variables affecting surfactant aggregate conversion in vitro. Exp Lung Res 1999; 25: 127-141 [Medline].

4. Reed JA, Ikegami M, Cianciolo ER, Lu W, Cho PS, Hull W, Jobe AH, Whitsett JA. Aerosolized GM-CSF ameliorates pulmonary alveolar proteinosis in GM-CSF-deficient mice. Am J Physiol 1999; 276: L556-L563 [Abstract/Free Full Text].

5. Whitsett JA, Glasser SW. Regulation of surfactant protein gene transcription. Biochim Biophys Acta 1998; 1408: 303-311 [Medline].

6. Korfhagen TR, Bruno MD, Ross GF, Huelsman KM, Ikegami M, Jobe AH, Wert SE, Stripp BR, Morris RE, Glasser SW, Bachurski CJ, Iwamoto HS, Whitsett JA. Altered surfactant function and structure in SP-A gene targeted mice. Proc Natl Acad Sci USA 1996; 93: 9594-9599 [Abstract/Free Full Text].

7. Elhalwagi BM, Zhang M, Ikegami M, Iwamoto HS, Morris RE, Miller ML, Dienger K, McCormack FX. Normal surfactant pool sizes and inhibition-resistant surfactant from mice that overexpress surfactant protein A.  Am J Respir Cell Mol Biol 1999; 21: 380-387 [Abstract/Free Full Text].

8. Beck DC, Ikegami M, Na CL, Zaltash S, Johansson J, Whitsett JA, Weaver TE. The role of homodimers in surfactant protein B function in vivo. J Biol Chem 2000; 275: 3365-3370 [Abstract/Free Full Text].

9. Korfhagen TR, Sheftelyevich V, Burhans MS, Bruno MD, Ross GF, Wert SE, Stahlman MT, Jobe AH, Ikegami M, Whitsett JA, Fisher JH. Surfactant protein-D regulates surfactant phospholipid homeostasis in vivo. J Biol Chem 1998; 273: 28438-28443 [Abstract/Free Full Text].

10. Wright JR. Immunomodulatory functions of surfactant. Physiol Rev 1997; 77: 931-962 [Abstract/Free Full Text].

11. Sano H, Sohma H, Muta T, Nomura S, Voelker DR, Kuroki Y. Pulmonary surfactant protein A modulates the cellular response to smooth and rough lipopolysaccharides by interaction with CD14. J Immunol 1999; 163: 387-395 [Abstract/Free Full Text].

12. van Rozendaal BA, Van de Lest CH, van Eijk M, van Golde LM, Voorhout WF, van Helden HP, Haagsman HP. Aerosolized endotoxin is immediately bound by pulmonary surfactant protein D in vivo. Biochim Biophys Acta 1999; 1454: 261-269 [Medline].

13. LeVine AM, Kurak KE, Wright JR, Watford WT, Bruno MD, Ross GF, Whitsett JA, Korfhagen TR. Surfactant protein-A binds group B streptococcus enhancing phagocytosis and clearance from lungs of surfactant protein-A-deficient mice. Am J Respir Cell Mol Biol 1999; 20: 279-286 [Abstract/Free Full Text].

14. Zhu S, Kachel DL, Martin WJ, Matalon S. Nitrated SP-A does not enhance adherence of Pneumocystis carinii to alveolar macrophages. Am J Physiol 1998; 275: L1031-L1039 [Abstract/Free Full Text].